Agarose gels for separation purposes have been commercially available for at least three decades. By derivatizing the basic gels—either in cross-linked or non-cross-linked form—the gels have been used in separations based on a number of different principles. Two well known product lines are Novarose (Inovata, Bromma, Sweden) and Sepharose (GE Health Care, Uppsala, Sweden).
Agar consists of about equal amounts of agarose and agaropectin both of which are polysaccharides with alternating anhydrogalactose and galactose subunits, i.e. their polysaccharide skeletons are the same. Agaropectin is significantly sulphated and therefore negatively charged. It is also methylated, i.e. contains methoxy groups. Agarose is in essence uncharged and without sulphate groups. Both agar, in particular desulphated agar, and agarose were initially suggested as starting materials for the manufacture of cross-linked separation gels. See for instance U.S. Pat. No. 3,959,251 (Porath et al). However during the years people have focused more on agarose than on agar, most likely due to the high content of sulphate groups of agar (present in agaropectin) and problems with removing the sulphate groups without negatively affecting the quality of the agar base material.
Conventionally agarose gels are obtained by cooling a warm solution of agarose to a temperature below its gelling temperature. The porosity of the gel will vary depending on the concentration of agarose in the starting solutions. A higher concentration will lead to a more dense gel (lower porosity) than a lower concentration. By including certain stabilising reactants in the solution the obtained gel will be stabilised, e.g. by cross-linking. The gel can be obtained in various physical forms such as flat bed, beads, plugs etc. Agarose gels in particulate forms have been obtained by emulsifying a warm solution of agarose in a solvent that is immiscible with water, cooling the solution below the gelling temperature of agarose, and collecting the particles. The sizes of the gel particles will depend on the sizes of the droplets in the emulsion that in turn will depend on stirring, emulgators etc. In practice the emulsifying process is studied in microscopy in order to decide when to stop the emulsifying step. Mostly a certain size fraction is desired which can be accomplished by sieving after the particles have been isolated. By including appropriate cross-linking agents (primarily water-soluble) in the emulsion the particles will be stabilized and the gelling temperature increased. The gel can be functionalised by introducing certain groups or ligands on the gel, e.g. affinity groups (including ion exchange groups) to make the gel suitable for affinity capture such as in affinity chromatography, enzymatically active groups to make the gel suitable for use in enzyme reactors, activated groups in order to enable introduction of any of the previously mentioned groups or as a support phase in solid phase synthesis etc.
Advantageous methods for producing rigid agarose separation gels from agarose and possibly derivatizing them are given in for instance U.S. Pat. No. 4,665,164 (Pernemalm et al), U.S. Pat. No. 4,973,683 (Lindgren G) and WO 1994004192 (Lindgren G).
There is a general desire to circumvent using highly purified and expensive agarose when manufacturing agarose separation gels, in particular cross-linked and/or derivatized forms of such gels. It would be attractive to have a simple method for producing them starting from less expensive material without loosing in performance characteristics compared to conventionally manufactured agarose separation gels.
All patents and patent applications cited herein are incorporated by reference in their entirety.